Project Summary/Abstract Metalloproteins perform chemical transformations with rates and selectivites that have yet to be achieved in synthetic or designed systems. These differences in reactivity are directly linked to the environment produced by the protein matrix that cannot be easily reproduced in synthetic constructs. To test our understanding of how metalloproteins function, we aim to design de novo metalloproteins from scratch. Proteins that bind porphyrin-like cofactors are of particular interest, as heme proteins are known to perform a variety of reactions. Only recently have we been able to design proteins that bind a cofactor with sub- accuracy. This opens the door to expand on the utility of natural proteins by incorporating synthetic inorganic cofactors into proteins that lack pre-evolved function. The proposed research strategy seeks to elucidate the design features necessary to bind M-tetraphenylporphyrin (M-TPP; M=Fe, Mn) complexes in close proximity to a substrate binding pocket. First- and second-shell interactions will be engineered to control orientation, electronic structure, and reaction pathway of the cofactor and substrate. Binding pockets will be designed for camphor and styrene and these de novo metalloproteins will be tested for their activity towards hydroxylation and epoxidation. The selectivity of these metalloenzymes will also be tested and the protein scaffold will be redesigned to elicit regioselective reactions (i.e. reacting with only one of two possible functionalizable groups). The proteins will be characterized using optical spectroscopies, electron paramagnetic resonance (EPR) spectroscopy, NMR spectroscopy, and by X-ray diffraction (XRD) methods. This work would be a breakthrough in protein design and will directly impact the fundamental understanding of the effects of protein environments on the function of metal centers in metalloproteins.